2.1. Glucose Consumption in Whole Blood
The phenomenon of in vitro glycolysis of whole blood has been recognized since 1876 (26,30). A specimen of whole blood exhibits a decreasing amount of glucose over time due to ongoing glucose consumption in vitro by the blood cells (26). As technology has improved, attempts at quantifying this rate have yielded results ranging from 1.9 mg % per hour (44) to 5-10 mg % per hour at room temperature (43) and from 5-10 mg % per hour (35) to 10-20 mg % per hour at body temperature (29).
In vitro glycolysis does not occur in nonhemolyzed serum (27), is temperature dependent (28,29), is independent of the presence of insulin and the initial blood glucose level (25), and depends both on the numbers of blood cells and on their metabolic activity. In vitro, leukocytes, erythrocytes and platelets all continue to metabolize glucose as an energy source.
Neutrophils use O.sub.2 to convert glucose to lactate through glycolysis, with hexokinase as the rate limiting step (38). A small amount, 2 to 3 percent, of the glucose metabolized is through the hexosemonophosphate shunt, the pathway that provides the NADPH needed to generate microbicidal oxidants and which pathway is "turned on" by phorbol myristate acetate (PMA). Lymphocytes also consume glucose, but with facilitated diffusion of glucose through the cell membrane instead of cell metabolic rate as the limiting step (39). Although the other white cell lines (monocytes, eosinophils, basophils) use glucose, the small numbers of these cells do not appear to impact significantly on the total amount consumed.
Erythrocytes use glucose through the glycolytic pathway and the hexosemonophosphate shunt. The metabolic dynamics and interactions are exceedingly complex, with many factors such as pH, lactate level, and accumulation of upstream metabolites having either stimulatory or inhibitory effects (40,41). PMA, however, does not change erythrocyte glucose utilization. Platelets use glucose depending on oxygen and glucose availability (42) and are stimulated by PMA.
In 1927, Falcon-Lesses showed that glycolysis rates were faster in whole blood from patients with leukemia than in normal blood (26). Since then, authors have described patients with leukemia (30), polycythemia (31,32) extreme eosinophilia (33) and leukocytosis (34) in whom this in vitro decrease in glucose concentration was so precipitous as to lead to very low glucose levels by the time of analysis--a condition called "artifactual hypoglycemia." On the other hand, Rawnsley and Bowman found that leukemic leukocytes consumed less glucose than normal leukocytes (35), suggesting that changes in leukocyte metabolism can also affect the amount of glucose consumed in vitro by a whole blood sample.
2.2. Neutrophil-Associated Host Autoinjury
Neutrophil-associated host autoinjury has been recognized as an important contributor to the endothelial damage that occurs in sepsis (1-9), adult respiratory distress syndrome or ARDS (5,10-13), multiple systems organ failure or MSOF (14-17), ischemia-reperfusion injury (18) and hemorrhagic shock (19). Neutrophils manifest a range of metabolic states--from normal quiescent cells (which derive their energy by anaerobic metabolism), to cells that have been primed by a variety of humoral mediators and that show an exaggerated response to a second activator (3), to fully activated cells. When activated, neutrophils undergo a series of marked metabolic changes in oxidative metabolism (the "respiratory burst"), characterized by a 50- to 100-fold increase in oxygen uptake, production of oxygen radical species (ROS), and the eventual degranulation of various neutrophil lysosomal enzymes (1,20,21). Primed neutrophils do not spontaneously display such activity, but show an enhanced response to activating stimuli with markedly augmented oxidative metabolism when compared to quiescent, non-primed cells. The neutrophil respiratory burst requires NADPH production, which is fueled by a large increase in glucose metabolism by way of the monophosphate shunt (20-22).
Respiratory burst capability, chemotaxis, spontaneous migration, chemiluminescence and direct superoxide anion production by neutrophils have been extensively investigated in sepsis (3,5). In particular, respiratory burst capability has been evaluated in sepsis, septic shock and longitudinally in septic shock (3,23). All these previous studies involved specialized analytical techniques using purified neutrophil preparations.
Circulating neutrophils become primed early in sepsis in the presence of circulating lipopolysaccharide and cytokines, including IL-1, TNF, IL-8, INF-.lambda., C5a, PAF and granulocyte colony stimulating factor. Compared to quiescent neutrophils, the additional stimulation of these primed neutrophils by either exogenous or endogenous agents causes many more of them to progress to respiratory burst. Paradoxically, neutrophils from patients who have progressed to septic shock show little response to additional exogenous stimulation (23), either because they are already maximally stimulated or because they have markedly depleted respiratory burst capability (1). It has been suggested that this diminished respiratory burst capability may be due to suppression by circulating humoral mediators (2). Serial neutrophil assays on patients who have survived septic shock have shown a return to normal function over time (2).
2.3. ROS as Markers for Neutrophil Metabolic State
The generation of ROS by neutrophils isolated from critically ill patients has been measured as a marker of neutrophil metabolic state. Evidence supporting in vivo priming of neutrophils during critical illness has been provided by measuring the generation of ROS by purified neutrophil isolates using either flow cytometry or spectrophotometry after an exogenous neutrophil stimulator (such as phorbol myristate 13-acetate (PMA) or N-formylmethionyl-leucyl-phenylalanine (FMLP)) is added (1,23). Opsonin receptor expression measured by chemiluminescence has also recently been used (24,25). These sophisticated laboratory techniques are time-consuming and expensive and require specialized expertise and equipment. Therefore, these methods are limited in their application to large scale use for either direct patient care or clinical research.
Hence, the determination of the metabolic state of a patient's neutrophils can provide invaluable information that is indicative of the condition of the patient and, in particular, may provide clues regarding the stage in which infection or disease has progressed. The amount of radical oxygen species generated by activated neutrophils, is obtained from purified neutrophil isolates requiring analytical techniques and sophisticated instruments not in general use. Thus, such techniques are severely limited and cannot be put into widespread use involving large numbers of patients. Furthermore, previous work done on in vitro glucose consumption in blood samples taken from ill patients had not established any demonstrable relationship between blood glucose consumption and neutrophil metabolic state. The difficulty of making measurements in whole blood is associated, in part, from the large number of cell types present in the blood which can affect the results of any measurement, as well as the large number of potentially interfering reactions taking place in the blood. In addition, certain phenomena, like the neutrophil respiratory burst, endure for only short periods, necessitating (as was widely held) purified cell preparations and sophisticated equipment and analytical techniques.
Thus, there remains a need for a method that can be made widely available to a great number of patients which provides information regarding the metabolic state of the neutrophils circulating in a patient's body. Such information can prove invaluable in the clinical setting and can lead to the identification of groups of patients who may best respond to a given treatment regimen. As described in greater detail below, with the information provided by the methods of the present invention, groups of patients manifesting clinical symptoms of non-sepsis, sepsis/severe sepsis, and patients in septic shock can be subcategorized further within each clinical group. With the substratification of such patients, those who may be at greatest risk or who may benefit the most from a particular prevention or treatment method may be identified expeditiously especially in those situations in which a proposed preventive/treatment regimen either is not widely accepted or requires a prophylactic or therapeutic agent that is not readily available.